There is a continuous need in medical practice, research and diagnostic procedures for rapid and accurate determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, seminal fluids and other biological fluids has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be detected (identified as a "ligand" herein) and receptors specifically reactive with that substance. Radioactive, fluorescent or enzyme labels have been used to detect the resulting reactive complex.
In recent years, the use of enzyme labels has received increasing attention because of various advantages over the use of radioactive and fluorescent labels. Assays using enzyme labels include what are known in the art as competitive enzyme immunoassays (EIA) and both direct and indirect enzyme linked immunosorbent assays (ELISA).
Another type of assay which has been developed is what is known in the art as an immunometric or a "sandwich" assay. Such an assay involves "sandwiching" the ligand (such as an antigen) with two or more receptor molecules (such as antibodies) which complex with the compound in a non-interfering manner and at different epitopic sites. Examples of such assays are described in U.S. Pat. No. 4,486,530 (issued Dec. 4, 1984 to David et al) where monoclonal antibodies having high affinity are used. In most sandwich assays, one or more of the receptor molecules are suitably immobilized on an insoluble carrier such as small particles, membranes, plates, or similar objects, and another receptor is suitable labeled, such as with an enzyme.
Peroxidase is one enzyme which has been used as a label in various assays, including specific binding assays of all types. Peroxidase acts on hydrogen peroxide as a substrate and can oxidize various chromogens or dye-providing materials to provide a detectable species in proportion to the amount of peroxidase present. Various dye-providing materials are known in the art, including benzidine and derivatives thereof. Such materials are described in U.S. Pat. Nos. 4,503,143 (issued Mar. 5, 1985 to Gerber et al) and 4,596,770 (issued June 24, 1986 to Parham et al).
In the latter reference, tetraalkylbenzidines are used in N-methylpyrrolidone as a dye-providing composition with peroxidase labeled antibodies in immunoassays. While such compositions may be useful if prepared and used immediately in a solution assay, it has been found that use of tetraalkylbenzidines provide insufficient sensitivity in certain assays where the ligand is present in the test fluid in low concentrations. Also the benzidine compositions are relatively unstable. In addition, in certain assays carried out using filter membranes, the membranes must be pretreated because the dye obtained from tetraalkylbenzidines, such as tetramethylbenzidine, is water-soluble and would otherwise pass through the membrane inadvertently.
It would be useful to have a more stable dye-providing composition which can be packaged into kit form for lengthy storage. It would also be desired to have a more sensitive diagnostic method which could be sold for use in various environments, including doctors' offices and in a consumer's home.